Characterization of a vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate

Vaccine 20 (2002) 2485–2493

Characterization of a vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate Ya-Ching Lin a,1 , Cheng-Nan Wu a,b , Shin-Ru Shih c , Mei-Shang Ho a,∗ b

a Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan c School of Medical Technology, Chang Gung University, Tao-Yuan, Taiwan

Received 20 November 2001; received in revised form 1 March 2002; accepted 11 March 2002

Abstract Enterovirus 71 (EV71) is a neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Because of the lack of an effective antiviral agent, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. Poliovirus vaccine technology, both live attenuated and inactivated, killed virus vaccines, can be adopted for use with EV71 because of their relatedness. In this study, we have characterized a laboratory-adapted EV71 strain, YN3-4a, which exhibits different characteristics from those of its parent isolate, neu, in having a rapid growth rate in Vero cells, a larger plaque size, and a lower LD50 in newborn mice. The YN3-4a can be produced at a high viral titer of up to 1010 tissue culture infective dose (TCID50 ) when grown in Vero cells, an approved substrate for virus vaccine production. Mouse antiserum raised against YN3-4a can neutralize a broad range of strains of EV71 isolated at different times from a variety of geographic regions. On passage in Vero cells, YN3-4a remained genetically and phenotypically stable. Many of the above-described features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage, and passage stability, are desirable features in a prototype virus for the development of an inactivated viral vaccine. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: EV71; Vero cell-adapted virulent strain; Inactivated virus

1. Introduction Since it was first identified 30 years ago, enterovirus 71 (EV71) has been implicated as the etiological agent in several large-scale outbreaks of severe neurological disorders in children throughout the world [1–5] and is now believed to be the most important neurovirulent enterovirus in the post-poliomyelitis eradication era [6]. In the recent years, not only a significant increase in EV71 epidemic activity has been noted throughout the Asia-Pacific region, a severe form of brainstem encephalitis with complications of pulmonary edema and high case fatality rates has been reported in these epidemics [7–11]. The development of vaccines for primary prevention is considered a top priority in order to reduce EV71-associated morbidity and mortality. Similarities in many of the virological and clinical aspects of poliovirus and EV71 strongly suggest that the vaccine strategy used against poliovirus, either live attenuated and inactivated ∗ Corresponding author. Tel.: +886-2-2789-9120; fax: +886-2-2782-3047. E-mail address: [email protected] (M.-S. Ho). 1 Present address: Fooyin Institute of Technology, Kaohsiung, Taiwan.

viral vaccine, could be effectively adopted to control EV71 infection. However, the concern of potential virulent revertant virus as that of oral polio vaccine has made the inactivated EV71 viral vaccine a more favorable choice for vaccine development. In a recent study, we demonstrated that mice receiving inactivated whole virus vaccine could mount a satisfactory level of antibody response that conferred protective immunity [12]. Proceeding with our effort in developing an inactivated viral vaccine, here we report our identification and characterization of a candidate prototype virus that possesses several desirable features suitable for use as an inactivated virus vaccine against EV71. 2. Materials and methods 2.1. Virus strains The primary EV71 isolate, neu, was obtained from an autopsy specimen of spinal cord from an 8-year-old child who died during the 1998 EV71 outbreak in Taiwan [13]. The autopsy specimen was first cultured with MRC5 cells to yield the primary viral isolate, which was then cultured in

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Vero cells. After two passages in Vero cells, plaque cloning was performed at an extremely low viral titer (multiplicity of infection, MOI = 0.0001) to obtain Vero cell-adapted strains. After a prolonged period of incubation (21 days), three plaques were obtained. Viruses derived from these plaques, hereafter referred to as the YN lineage, were further passaged in Vero cells and were found to have different phenotypes from that of the parent virus, neu (Fig. 1). Other test strains of EV71were all viral isolates collected from infected children who either died or manifested severe neurological symptoms during the EV71 outbreaks in

1998 and 2000 in Taiwan and during the 1997 outbreak in Malaysia [5]. This collection of viruses is presumed to represent a variety of viral strains causing mortality and severe morbidity in outbreaks with a quite wide geographic and temporal span. 2.2. Cell culture and viral proliferation Vero cells (ATCC CCL81, green monkey kidney cells) were routinely maintained in Dulbecco’s modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS).

Fig. 1. Origin and history of passages of the YN and neu lineages of enterovirus 71. All viruses of the YN lineage were given a numerical designation following the dash. The number following the first dash designates the plaque, and the numbers following the second and third dashes represent the passage number.

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For routine viral propagation, a monolayer of Vero cells (∼80% confluence) was infected with virus at a MOI of 0.1–0.5 in DMEM containing 2% FBS and incubated at 37 ◦ C in 5% CO2 . When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the virus was harvested by two freeze-thaw cycles of the infected cells in culture medium, cellular debris being removed by centrifugation at 2000×g for 6 min. The fourth passage of YN3-4a, YN3-4a-4, was largely stocked for following experimental use. 2.3. Viral titration Viral titer was determined as the 50% tissue culture infective dose (TCID50 ) in Vero cells. Monolayers of Vero cells (104 cells/well) in 96-well plates were inoculated with serial ten-fold dilutions of virus and viral growth monitored by microscopic observation for typical enteroviral CPE. On the fourth day, the TCID50 ml−1 was determined by the Reed and Muench method [14] as the reciprocal of the lowest viral dilution that resulted in CPE in more than 50% of cells in the well. 2.4. Plaque assay The plaque assay was performed in 6-well plates. Virus (serial ten-fold dilutions in serum-free DMEM) was allowed to adsorb to confluent monolayers of Vero cells (2 × 106 cells/well) for 1 h at 37 ◦ C, then the inoculum was removed before overlay with 3 ml of 0.8% purified agar (Noble, Difco) in DMEM, 5% FBS and incubation of the plates for 7 days at 37 ◦ C in 5% CO2 . The viral inoculum was pre-treated with chloroform to prevent clumping of the virus and to improve plaque formation [15]. 2.5. Viral growth assay Confluent monolayers of Vero cells (105 cells/well) in 24-well plates were infected with virus at an MOI of 0.01, 0.1, 1, or 5. The virus was allowed to adsorb for 1 h at 37 ◦ C, then the cells were grown in 500 ␮l of DMEM, 2% FBS. For time-course experiments, multiple identical wells were set up, then, at each time-point, the contents of one well were harvested, the culture medium and cells, resuspended in the original volume of culture medium, being collected separately and stored at −70 ◦ C for the subsequent determination of the extracellular and intracellular viral titers. All viral titers were determined in one batch by the TCID50 , as described above. 2.6. Neutralization test The serum neutralization titer was determined using the 50% TCID50 reduction assay for viral neutralization in Vero cells [16]. Antisera were obtained from female BALB/c mice immunized intraperitoneally (i.p.) at 4–6-week-old with two

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doses (each 10 ␮g of protein, separated by 4 weeks) of either neu or YN3-4a virus that had been purified with ultracentrifugation and inactivated as previous described [12]. Prior to assay, the antisera were heat-inactivated at 56 ◦ C for 30 min and serial two-fold dilutions prepared. The diluted samples were then incubated at 4 ◦ C overnight with an equal volume of DMEM containing a viral dose of 100 TCID50 , then the virus/antiserum mixture was added to a monolayer of Vero cells in 96-well plates (104 cells/well). After incubation for 1 h at 37 ◦ C, the medium was removed and the cells incubated in 100 ␮l of fresh DMEM, 2% FBS. The titer of the antiserum was determined as the highest dilution which was able to inhibit CPE in >50% of the cells in the well. All samples were tested in triplicates; if the difference between triplicates was greater than two-fold, the assay was repeated. Coxsackievirus A16 was included as a reference cross-reactive serotype that sets the boundary between EV71 and other non-EV71 enteroviruses. 2.7. Virulence assay The virulence of the neu and YN strains was tested in newborn ICR mice. The viral inoculum, at doses ranging from 102 to 106 TCID50 in a volume of 100 ␮l, was inoculated i.p. into groups of mice (2 l of newborn mice per dose of inoculum) and the mice were observed daily for signs of hind limb paralysis and death. The LD50 was determined by the method of Reed and Muench [14]. 2.8. DNA sequencing Viral RNA was extracted from the culture medium using a commercially available kit (QIAamp viral RNA mini kit, Qiagen Inc., Santa Clara, CA). Sequencing analysis of the full-length viral genome was carried out by direct sequencing of the RT–PCR products, as previously described [8]. Analysis of the 5 - and 3 -untranslated regions (UTR) was carried out by adding a nucleotide sequence tag to the ends of the full-length viral cDNA (5 Race and 3 Race systems, GIBCO BRL Products). This sequence tag served as the target for primer annealing during PCR amplification and automatic sequence analysis (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin-Elmer Inc., CA).

3. Results 3.1. Origin of the YN lineage After being grown in MRC5 cells, then in Vero cells, the clinical isolate strain, neu, showed a rapid reduction in viral titer from 1010 to 102 TCID50 ml−1 by the fourth passage. At a very low MOI of 0.0001 in Vero cells, only three plaques were generated after 21 days of incubation. Further passage of the three plaques in Vero cells yielded the YN lineages,

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Table 1 Maximal viral titers of various virus strains grown in Vero cells at an MOI of 1 Virus strain Maximal viral YN3-4a-2 YN4-2 YN5-2 YN3-3 neu5 a

Intracellular titera

(TCID50

Extracellular

ml−1 ) 2.5 6.4 2.0 2.0 1.0

× × × × ×

109 107 107 107 106

1.0 1.0 3.2 3.2 3.2

× × × × ×

106 106 105 105 105

Data extracted from the peak of viral production in the growth curve.

YN3, YN4, and YN5 (Fig. 1). The YN lineage viruses differed from the parental neu virus in their remarkably productive infection in Vero cells (Table 1). A virus showing particular rapid growth in Vero cells was derived by harvesting the virus released into the culture medium within 24 h of inoculation with YN3-4 (MOI = 1); this virus was termed YN3-4a. The high viral titer of YN3-4a could be reproducibly maintained during further passages, therefore YN3-4a became the focus of this study for further consideration as a vaccine candidate.

growth kinetics to YN3-4a, but the maximal viral yield was 1000-fold less, i.e. 108 versus 1011 (Figs. 2d and f). Furthermore, YN3-4a and neu differed in plaque morphology in Vero cells. The plaques formed by YN3-4a were heterogeneous in size, some being as large as 4 mm in diameter and others only 1 mm in diameter (Fig. 3B); in contrast, the parental neu strain only produced small plaques of 1 mm diameter. The mixed size plaque morphology was still seen after chloroform pre-treatment of the virus. 3.3. Sequence analysis Comparative sequence analysis of the entire viral genome for YN3-4a and the parent neu strain showed 95.4% identity of nucleotides. The most variable region was in region 3C, which showed 92% identity of nucleotides and only 87% identity of amino acids (Table 2). The VP1 region showed only 87% identity of nucleotide sequence, but 96% identity of amino acids. When the untranslated regions (UTR) were compared, the two strains showed 98% identity of nucleotides at the 5 UTR, but only 92% at the 3 UTR. The longest poly-A tail in neu was 89 bases in one clone, while the majority of YN clones had a 54-base poly-A tail.

3.2. Growth kinetics 3.4. Virulence The growth properties of YN3-4a were compared in Vero cells with those of the parental strain, neu5 (the fifth passage of neu), at MOIs of 0.1, 1, and 5 (Fig. 2). The yields of released and cell-associated virus were studied separately. Overall, YN3-4a showed a significantly higher viral yield than neu at all time intervals at all three MOIs. Both neu and YN3-4a yielded more intracellular viral particles than released virus. With the exception of neu at an MOI of 0.1, under all growth conditions, the amounts of the released and intracellular forms of both YN3-4a and neu reached a peak, then fell, indicative of decay of viral viability. The time interval to reach the peak viral titer was inversely proportional to the MOI for each growth condition and was generally shorter for YN3-4a than for neu, e.g. using an MOI of 5, the peak occurred at 16 and 20 h post-infection with YN3-4a and neu, respectively (Fig. 2e and f). At a lower MOI infection (0.1), the intracellular and extracellular titers of YN3-4a peaked at 30 and 40 h post-infection, respectively, then declined (Fig. 2a), whereas neu showed very retarded growth kinetics that did not reach a peak within the time interval studied (Fig. 2b). At MOIs of 1 and 5, neu showed similar

The virulence of the YN3-4a strain in newborn ICR mice was compared to that of the parental neu strain. Intraperitoneal inoculation of newborn mice with the YN3-4a strain at a titer of 103 or 104 TCID50 ml−1 resulted in 100% mortality, the mice dying within 5 days after the appearance of severe hind leg paralysis. The LD50 of the YN strain was 2.3 × 101 TCID50 ml−1 (Fig. 4b), whereas that of the neu strain was 106 TCID50 ml−1 (Fig. 4a). 3.5. Ability to grow in serum-free medium The ability of YN3-4a to proliferate in serum-free medium was studied. Monolayer Vero cells were infected with YN3-4a, then, after adsorption, the FBS-containing medium was replaced with medium containing B-27 serum supplements, and incubation continued under the conditions described in Section 2. The two culture systems were found to be equally effective in supporting the growth of YN3-4a to a comparable high titer and with similar growth kinetics (Figs. 5a and b).

Table 2 Identity (%) of nucleotide and amino acid sequences of the viral genomes of YN3-4a and neu 5 UTR

VP4

% identity between YN3-4a and neu Nucleotide 98 99 Amino acid – 100 UTR: untranslated region.

VP2

VP3

VP1

2A

2B

2C

3A

3B

3C

3D

3 UTR

99 99

98 97

86 96

99 100

98 97

94 93

95 94

97 95

92 87

97 97

92 –

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Fig. 2. Growth kinetics of viruses in Vero cells infected at various multiplicities of infection (MOI). Viral titers in the culture medium ((䊉), extracellular titer) and in cells resuspended in the original volume of medium ((䊊), intracellular titer) were determined separately. Viral production is shown at MOIs of 0.1 (a and b), 1 (c and d), and 5 (e and f).

3.6. Antigenicity and antigenic breadth Since YN3-4a seemed to represent a possible vaccine candidate, it was important to determine whether it retained the critical antigenic epitopes needed to generate antibodies able to neutralize a broad spectrum of wild strains of EV71. When equivalent amounts of viral protein were used to immunize BALB/c mice, YN3-4a persistently induced a higher level of autologous neutralizing antibody than the neu strain (titers of 2560 and 640, respectively; Table 3). In addition to the quantitative difference in the induction

of autologous antibody, antiserum raised against YN3-4a also demonstrated a better heterologous antigenic coverage against most viruses tested than that raised against the neu strain. When the titers of both autologous antisera were adjusted to an equal titer of 640, antiserum raised against YN3-4a consistently exhibited a higher (≥two-fold) heterologous neutralizing antibody titer for seven of the 15 viruses tested than that raised against neu (Table 3). These data indicate that immunization with YN3-4a resulted in the efficient production, both quantitatively and qualitatively, of neutralizing antibodies against a broad range

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Fig. 3. Plaque morphology of neu (a), and YN3-4a (b).

of strains of EV71 (P = 0.05, Wilcoxon’s signed rank test). 3.7. Stability As a result of adaptation during prolonged tissue culture, viruses may change their genetic properties with time. To check the genetic and phenotypic stability of the YN3-4a lineage grown in Vero cells, we selected the sixth (YN3-4a-6), ninth (YN3-4a-9), and twelfth (YN3-4a-12) passages of YN3-4a grown in Vero cells for further analysis. The growth kinetics was near identical for all four passages (Fig. 6). Analysis of the genetic sequences of the viral VP1, 3C, 5 and 3 UTR regions, the regions most likely to show genetic variation, showed the VP1 and 3C amino acids to be identical in all passages (Table 4). The 5 UTR nucleotide

Fig. 5. Growth kinetics of the YN strain in Vero cells (MOI = 1) in serum-containing and serum-free medium. (a) DMEM medium containing 2% FBS; (b) DMEM containing B-27 serum supplement. Total, (䊏); extracellular, (䊉); and intracellular, (䊊); viral titers were expressed as TCID50 ml−1 .

sequences showed 98% identity with that of YN3-4a for all passages, and those of the 3 UTR were identical in all passages. Moreover, when antigenic stability after different number of passages was evaluated by cross-neutralization using antisera prepared against the YN3-4a-6, YN3-4a-9,

Fig. 4. Survival rate of newborn ICR mice infected with enterovirus 71. (a) Mice inoculated intraperitoneally (i.p.) with the neu strain at a dose of 106 TCID50 ml−1 , the highest viral titer that could be used; (b) mice inoculated i.p. with YN3-4a strains at doses ranging from 102 to 106 TCID50 ml−1 .

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Table 3 Neutralization of various wild types of enterovirus 71 by antisera from mice immunized with the YN3-4a and neu strains

Table 5 Cross-neutralization test of the passages of YN3-4a virus by antisera derived from these passages

Virus strains

Virus tested

Neutralization titer of YN3-4a antiserum

YN3-4a 2560 (640)a neu6 640 (160) Der 1280 (320) 1743(B) >10240 (2560) 1997/1998 Malaysia isolates 14343 Throat 640 (160) 13898 Medulla 2560 (640) 13898 Throat swab 1280 (320) 13898 Spinal cord 320 (80) 13898 Stools 320 (80) 13091 Brain 320 (80) 14245 Throat swab 160 (40) 16985 Stools 320 (80) 1998 isolates from central Taiwan 5277 Vesicle swab (OP) 320 (80) 5414 Throat swab 640 (160) 5415 Stools 640 (160) 2000 isolates from Taipei Taiwan 2 K 0652 320 (80) 2 K 1148 1280 (320) Cox A16 80

neu5 Antiserum YN3-4a-4 YN3-4a-6 YN3-4a-9 YN3-4a-12

40 640 160 320 40 160 160 160 160 80 40 40

Antiserum produced using YN3-4a-4

YN3-4a-6

YN3-4a-9

YN3-4a-12

640 640 320 640

320 640 320 320

320 320 640 320

320 320 320 640

and YN3-4a-12 passages, all the antisera reproducibly neutralized all the test viruses with similar efficiency (Table 5).

4. Discussion

160 160 160 40 80 40

a

Adjusted titers, based on the adjustment of the titer of the autologous antserum to 640.

Fig. 6. Comparison of growth curves of YN3-4a at the fourth, sixth, ninth, and twelfth passages in Vero cells infected at an MOI of 1.

The two conventional viral vaccine technologies, inactivated and attenuated virus vaccines, have an advantage over the newer subunit vaccine technology in that their regulatory standards are well established, making it much simpler to streamline research and the development and licensing of vaccines to meet immediate demands for disease control. Attenuated viral vaccines carry a certain risk of adverse effects due to virulent revertants, which have always been a concern in the use of live attenuated poliovirus vaccine [17–19]. Even with the yellow fever vaccine, which has a long-standing good safety record, virulent mutants occasionally cause mortality [20]. Although, it has an advantage in terms of safety, inactivated virus vaccine is less immunogenic and a much higher dose of viral antigen is required to achieve an acceptable level of immunogenicity and protection. Thus, the identification of viral strains that can grow to a high viral titer in host cell lines that are approved for vaccine production represents the first step in vaccine development. In this report, we describe a laboratory-adapted EV71, YN3-4a, which can grow well in Vero cells, an approved cell line for vaccine production. YN3-4a can reach a peak titer of 1010 to 1011 TCID50 ml−1 when grown in Vero cells. Moreover, it can be propagated in serum-free culture medium and achieve a similar high titer. Due to concerns about contamination with bovine viral diarrhea virus and the induction of antibody against fetal calf serum components in the viral growth medium [21–23], the ability to propagate the virus in serum-free medium is a desirable feature in vaccine production.

Table 4 Homology (%) of nucleotide and amino acid sequences in the VP1, 3C, 5’UTR, and 3 UTR regions of YN3-4a and its subsequent passages Passage

% Homology YN3-4a-6 YN3-4a-9 YN3-4a-12

VP1

3C

Nucleotide

Amino acid

Nucleotide

Amino acid

99 99 99

100 100 100

100 100 100

100 100 100

5’UTR (nucleotide)

3’UTR (nucleotide)

98 98 98

100 100 100

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The growth kinetics for both the released and intracellular virus peaked, then declined, a phenomena making it difficult to determine the most optimal time for virus harvesting. Experiments were performed at a low MOI (0.01) that made it possible for multiple harvesting of the released virus every 48 h for up to three times (data not show). Using this modification, it was possible to achieve a total released viral titer of 1.8 × 108 TCID50 ml−1 per culture batch thus resolving the need to purify intracellular viral particles, thus simplifying the procedure for vaccine production. Immunization of mice with the YN3-4a strain induced a high titer of antibody that could neutralize a wide spectrum of EV71 strains in vitro, suggesting that the antibodies produced by YN3-4a possess a broad antigenic cross-reactivity and neutralizing ability against all enteroviruses tested. Compared to the parental neu strain, YN3-4a was quantitatively more immunogenic, resulting in a higher titer of autologous neutralizing antibody, and the antiserum possessed a qualitatively broader coverage, i.e. it had a higher heterologous neutralizing antibody titer. Genetic analysis of YN and neu indicated that the major differences that might contribute to the antigenic diversity are probably localized in the VP1 region, in which the immunodominant antigenic epitopes that are independent of other capsid viral proteins are probably located [12]. It has been reported that a single amino acid substitution in the VP1 protein of Coxsackievirus B4 can alter its antigenicity and virulence [24–26]. Exactly how the 14% nucleotide and 4% amino acid sequence differences between VP1 in YN3-4a and neu might contribute to the differences in antigenicity, antigenic breadth, and virulence in newborn mice remains to be determined. The growth rate of the YN3-4a strain differed significantly from that of the neu strain, with the highly virulent YN3-4a strain growing more efficiently, suggesting that improved growth might contribute to the viral virulence. Studies of attenuated simian immunodeficiency virus vaccine have indicated that the degree of protection against AIDS infection depends on the replication capacity of the vaccine strain [27–30]. Evaluation of the vaccine efficacy of HIV suggested that failure of the vaccine to protect from infection was related to lower viral virulence [31–33]. Such a paradigm in the correlation of the growth capacity, virulence, and vaccine efficacy offers an important point to consider for the development of an inactivated viral vaccine. While the virological bases for the immunogenic advantage of YN3-4a over neu remains elusive, it is crucial that the YN lineage, if used as a prototype viral vaccine, should remain genetically stable with further passages in Vero cells. Results of the direct DNA sequencing analysis of RT–PCR product of viral genes indicated that Vero cells did not significantly exert further selection pressure in favor of a particular viral quasi species that might have arisen in subsequent passages. The stability was further supported by the phenotypic studies of growth kinetics and antigenic nature of YN lineage in Vero cells. While the biological and molecular basis for, and the clinical significance of, the many unique features of the

YN lineage remain to be delineated, the characteristics of YN3-4a described in this report, support the notion that it could serve as a prototype inactivated vaccine candidate. Its high growth rate contributing to an increased yield of virus in serum-free medium is an advantages in vaccine production that is especially crucial for inactivated viral vaccine. Moreover, the neutralization results reveal that this vaccine candidate is highly immunogenic and also that the YN3-4a virus-induced antibodies neutralize various wild-type EV71 viruses. The YN3-4a virus therefore appears to be a promising candidate as an inactivated EV71 vaccine.

Acknowledgements We thank Drs. Kenneth Sai Kit Lam and Chung Liang Kao, who kindly provided us with various enterovirus 71 strains, and Drs. Chin-Yuan Lee, Min-Yi Liao, Wen-Li Lin, and Yi-Lin Lin for their inspiring discussions and helpful suggestions during the conduct of this study. Grant support: (1) an institute grant of the Academia Sinica 1999, (2) Taiwan Center for Disease Control Grant #CDC89-VCRD-003. References [1] Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 1974;129:304–9. [2] Chumakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol 1979;60:329–40. [3] AbuBakar S, Chee HY, Shafee N, Chua KB, Lam SK. Molecular detection of enterovirus from an outbreak of hand, foot and mouth disease in Malaysia in 1991. Scand J Infect Dis 1999;31:331–531. [4] Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. New Engl J Med 1999;341:929–35. see comments. [5] AbuBakar S, Chee HY, Al Kobaisi MF, Xiaoshan J, Chua KB, Lam SK. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res 1999;61:1–9. [6] da Silva EE, Winkler MT, Pallansch MA. Role of enterovirus 71 in acute flaccid paralysis after the eradication of poliovirus in Brazil. Emerg Infect Dis 1996;2:231–3. [7] Ng DK, Law AK, Cherk SW, Mak KL. First fatal case of enterovirus 71 infection in Hong Kong. Hong Kong Med J 2001;7:193–6. [8] Shieh WJ, Jung SM, Hsueh C, Kuo TT, Mounts A, Parashar U. Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan. Emerg Infect Dis 2001;7:146–8. [9] McMinn P, Stratov I, Nagarajan L, Davis S. Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis 2001;32:236–42. [10] Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin Infect Dis 2000;31:678–83. [11] Wong KT, Lum LC, Lam SK. Enterovirus 71 infection and neurologic complications. New Engl J Med 2000;342:356–8.

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